The present invention relates generally to the field of mid-infrared (mid-IR) spectrometry, and more specifically to an attachment to infinity-corrected, commercially available light microscopes to provide the techniques of internal and external reflection infrared microspectrometry.
Spectroscopic analysis using radiant energy in the infrared region of the electromagnetic radiation spectrum is a primary technique for chemical analysis of molecular compounds. The infrared spectral region extends from 0.7 to 250-micrometers, however the mid-IR region is generally considered to cover the region from about 2.5 to about 25-micrometers (or parts thereof), which is commonly used for molecular vibrational spectroscopy. While the primary distinction between near-IR and mid-IR regions is based upon whether the underlying molecular frequencies are fundamental or overtone frequencies, instrument components tend to differ and also be specific by region. There is some overlap however, and specifically mid-IR Fourier transform infrared spectrometers typically cover that part of the near-IR region from 1 to 2.5 micrometers.
This invention defines an attachment apparatus and method for infrared spectroscopic or radiometric analysis of microscopic samples of solids or liquids, including biological materials, combining external or internal reflection spectroscopy with visible light and near-IR radiant energy viewing of microscopic samples by using an attachment to standard commercially available visible light microscopes and commercially available video cameras. The magnification optics for infrared spectral analysis are infrared transmitting objective lenses that are used to focus a beam of radiant energy onto a sample, or sample surface, collect the reflected radiant energy, and present that energy to a detector system for spectral analysis.
Since the introduction of commercial infrared microspectrometers, the advantage of combining the capabilities of a visible-light microscope with an infrared spectrometer has been of great importance. Infrared microscopes, such as those disclosed in U.S. Pat. No. 4,878,747 (the ""747 patent) issued to Donald W. Sting and Robert G. Messerschmidt, have been used for an ever-expanding range of applications. These specialized microscopes were attached to commercial Fourier transform infrared (FT-IR) spectrometers. Such microscope/FT-IR systems have been used to detect and identify trace contaminants, to analyze multilayered composites, micro-electronic devices, phase distributions in polymeric materials, inclusions in minerals, abnormal cellular materials, DNA, and numerous other materials.
Heretofore, all known combinations of mid-IR spectrometers and visible light microscopes were composed of (1) a combination of a general purpose laboratory spectrometer and an attachment to the spectrometer having visible light illumination and viewing, or (2) a specially designed integrated instrument combining infrared spectroscopy and visible imaging features. In all cases, the resulting products emphasized the infrared spectroscopy capability, utilizing visible microscopy capabilities as a means to support the infrared spectroscopy capability.
Known special infrared microscope systems and attachments to mid-IR spectrometers have become pervasive even though such systems and attachments are costly and complex. The microscope attachments to laboratory FT-IR spectrometers, described in the ""747 patent to Sting and Messerschmidt, among others, have become the standard configurations for infrared microspectroscopy systems. These complex microscope attachments typically provide both transmission and reflection capabilities and use variable remote-image-plane masks to define sample areas for infrared analysis. All of this known art, however, consists of special purpose FT-IR microscopes with specialized optical systems that are appended to large bench-top spectrometers, or fully integrated FT-IR microscope systems using some visible light microscope components. No such systems known use an attachment to visible light microscopes, as is contemplated by our invention.
Our invention provides for the use of both external-reflection and internal-reflection microspectroscopy techniques. Internal-reflection microspectroscopy provides certain advantages over both transmission and external reflection microspectroscopy, particularly in the ability to analyze thick samples. With the introduction of internal-reflection microspectrometry, as shown in U.S. Pat. No. 5,093,580 to Donald W. Sting, and U.S. Pat. No. 5,200,609 to Donald W. Sting and John A. Reffner (also known as attenuated total reflection microspectrometry or micro-ATR) reflection microspectrometry has gained ever-greater importance. Furthermore, our invention extends the capabilities of internal-reflection microspectroscopy by using the unique ATR technology disclosed in U.S. Pat. Nos. 5,703,366 and 5,552,604 issued to Sting and Milosevic to create a novel infinity-corrected ATR objective used for microspectroscopy.
All previous forms of infrared microspectroscopy apparatus were designed from the perspective of the spectroscopist, whereas this invention is designed from the perspective of those using visible light microscopes. Our invention treats the infrared spectroscopy capability as an adjunct to a visible light microscope, and thereby provides extension of the visible microscope""s capabilities. It is a primary object of the present invention to provide an FT-IR spectrometer attachment that is easily attached to a commercially available light microscope without compromising any of the available visible light microscope features, options, and capabilities.
The present invention provides an optical system, apparatus and method to use a mid-IR spectrometer system as an attachment to commercial light microscopes for molecular analysis of materials. In this invention a small spectrometer, in combination with optical, mechanical and electronic components, form an apparatus that can be directly attached to a light microscope for measurement of infrared spectra of microscopic samples or sample domains. Because it can be readily attached directly to existing microscopes, using conventional mechanical connectors that are typically used for microscopes, costs are significantly lower than the current art method of using a dedicated infrared microscope that is attached to a laboratory FT-IR spectrometer. Furthermore, because of the ease of use and accessibility of such low cost infrared spectroscopy capability to material scientists, biologists, and pathologists, as well as others using conventional visible light microscopes, it is expected that significant interdisciplinary benefits will occur.
Using our invention, infrared spectra are acquired using either the external-reflection or the internal-reflection spectroscopy technique. By using reflection spectroscopy techniques, nearly all types of samples can be analyzed. A thin film of material for example, can be mounted on an infrared reflective, but visibly transmissive, substrate such as low-E glass to be analyzed by reflection-absorption, a special case of external-reflection, whereby infrared radiation from the spectrometer is directed onto and through the sample film to the low-E glass substrate, where the radiation is reflected and subsequently passes through the film a second time, whereupon the radiation ultimately is directed to a detector for analysis. An absorption spectrum is thereby acquired, but the measurement was made using the external-reflection technique. For external-reflection spectroscopy, the external-reflection infrared objective lens does not contact the sample, as it must with the ATR objective lens which is used for internal-reflection spectroscopy.
Any thick or thin sample that is placed in contact with the internal-reflection element of an ATR objective lens can result in an ATR spectrum. Because the infrared spectrum of most samples can be measured by using either internally or externally reflected radiation, the infrared spectrometer attachment can provide molecular analyses in a simple and economical manner.
Another object is to use infinity-corrected reflecting objectives and complementary optical components both to direct radiant energy onto a microscopic area and to allow visualization of the magnified image of the specimen and of a highly correlated measure of the mid-IR radiation. The near-IR radiation from the infrared source is used to get this magnified image and correlated measure through an integral video system. Visualization of the mid-IR radiation is achieved by bringing together three distinctly separate ideas in a novel way. First, infrared spectrometers, and specifically mid-IR FT-IR spectrometers, provide a source of infrared radiation that includes some near-IR radiation. Second, commercially available video camera arrays are sensitive to near-IR radiation. Finally, commercially available optical elements are readily made that transmit or reflect radiation differently for different wavelength regions. Using these facts in a novel way caused us to define a new term, a xe2x80x9ctrichroicxe2x80x99 element, meaning an optical element with defined functions in three different wavelength regions. For example, in the preferred embodiment of the mid-IR attachment, the trichroic element largely transmits visible light radiation, it both transmits and reflects near-IR radiation, and it largely reflects mid-IR radiation. The specifics of how the trichroic element is used in conjunction with the preferred embodiment is discussed in detail when describing FIGS. 7 and 8. Using this novel idea and others has allowed us to incorporate the mid-IR spectrometer attachment into infinity-corrected light microscopes to provide unique and significant benefits to the microscopist. In all embodiments, the inclusion of the mid-IR attachment on the microscope maintains a simple optical system without compromising any of the standard features and capabilities of the light microscope.
A still further object of the present invention is to provide a simplified system for detecting contact of a sample with an internal reflection element. Observing physical contact with visible light is a direct indication of optical contact in the mid-IR region since visible region wavelengths are shorter than mid-IR region wave lengths. That is, if contact is achieved at a shorter distance (less than a quarter of a visible wavelength) it must be achieved for the longer mid-IR region wavelengths.
One embodiment of the invention provides an optical system, which meets the Koehler illumination criterion of focusing the source element of the radiation at the pupil (aperture) of the objective lens. Visible light illumination systems typically meet this criterion, and this embodiment of our invention meets the Koehler illumination criterion for both visible and infrared radiation. To our knowledge, infrared microspectrometer systems have never before been designed to meet the Koehler illumination criterion. This embodiment of our invention, which meets this criterion, we believe, will be of increasing importance to infrared microspectrometry as infrared array detectors become more readily available at affordable prices.
Other objects of this invention will be apparent from the following description, which is provided to enable any person skilled in the art to make and use the invention, and which sets forth the best mode contemplated by the inventors of carrying out their invention. Various modifications to the specific embodiments disclosed herein, within the general principles of the invention as defined herein, will be apparent to those skilled in the art.